Cryopreservation has been the main method for preserving biological specimens for many years. It has allowed researchers to use rare or precious samples from decades ago to answer new research questions. Today its used to preserve the latest complex cell models for other scientists to use in the future and is essential for the storage and supply of biological treatments. In this article, we explore the challenges with cryopreservation and the emerging advances that could improve these methods.
There are a few ways of achieving this: the most common approach used in research labs is to freeze samples at 80 C using solid CO2 or 196 C using liquid nitrogen. But a method called vitrification is used for freezing clinical samples such as sperm, fertilized eggs or ovarian tissue for long-term storage. The main difference is that traditional cryopreservation methods allow ice to form during the preservation process whereas in vitrification the whole solution is solidified without any ice crystallization.1 In this article we will focus on conventional cryopreservation most typically used in labs.
Without cryopreservation you must keep cells and tissues alive in continuous culture which means growing and splitting them to generate more cells (called passaging). But cells change as they multiply over time, and this can cause them to lose important characteristics. By freezing them, it reduces the heterogeneity which would otherwise be introduced by passaging them repeatedly.
From our perspective, cryopreservation allows us to bank new cellular models for the research community which people can go back to for years to come, says Dr Charlotte Beaver, a senior scientific manager at the Wellcome Sanger Institute who develops complex cell model systems such as organoids. It means we can keep the model long term, but they're not dividing continually to the point where they've mutated to barely represent the cells you started with.
One area where cryopreservation is becoming increasingly important is in the rapidly emerging field of cell-based therapies: for example, preserving mesenchymal stem cells for a transplant, or chimeric antigen receptor T cells (CAR T cells) for treating cancer. With CAR-T cell therapy, a patients T cells are removed, re-engineered to recognize antigens (such as tumor-associated antigens) and then returned to them. You've got to do a lot of processing on these cells, potentially moving the cells between sites, getting them from the patient when they donate them to the lab, and then back to the patient again, and the cells can degrade really easily, explains Professor Matthew Gibson, a biomaterial scientist at the University of Warwick. Treatments like these ideally need freezing in a format that lets you quickly thaw them at the bedside and then administer them to patients. As soon as you have to do lots of processing in a hospital setting, that creates a barrier and all these steps introduce a large cost. These are extremely expensive therapies anyway, so anything to make that process more efficient and results in getting more healthy cells back after freezing is good for the patient.
Cryogenically Storing Animal Cell Cultures
Maintaining healthy, growing cell cultures is a demanding task made more difficult by the ever-present risk of their loss through accidents or contamination. These problems can be reduced by using cryogenic preservation, a process that effectively puts the cells into true suspended animation. Download this guide to discover intracellular and extracellular events during cell freezing and the advantages of freezing cell cultures.
Sponsored Content
During the freezing process, ice crystals form within the sample and damage the cells and some of the cells never recover. Most lab and clinical protocols use cryoprotectants to protect the cells from ice crystals and/or controlled rate freezing and thawing to avoid shocking the cells with sudden changes in temperature.Many different cryoprotectants are available, but the most common cryoprotectant used for mammalian cells and tissues is dimethyl sulfoxide (DMSO). Other options include glycerol for bacterial cells and red blood cells. But the cryoprotectants have their own drawbacks; DMSO is considered the best protectant but at certain concentrations it is toxic to cells. Glycerol is kinder to cells but less effective as a cryoprotectant compared with DMSO.2 One way to mitigate potential toxicity problems is to use different combinations of cryoprotective agents, such as supplementing a lower concentration of DMSO with glycerol or polypropylene glycol. 2There are also some primary cell types that just don't like being frozen in cryoprotectant solutions, which can present a challenge. Acute myeloid leukemia peripheral mononuclear cells are much easier to freeze and recover than lymphoma cells, so if you are freezing samples where you dont yet know what the malignancy is then you need to treat them very carefully, explains Iqbal. Equally, different types of healthy blood cells freeze differently T cells may recover well, although their functionality can be compromised, whereas granulocytes will not recover at all.
With novel complex models like organoids derived from a patient sample, we then have the danger of losing models, explains Beaver. One of the advantages of using organoids is that they are comprised of different cell populations representing the natural heterogeneity that you see within a tissue or tumor. Freezing puts them through a bottleneck and if that bottleneck is too harsh for some of the cells, you lose that heterogeneity and it's not such a good model anymore.
Another consideration is the end use of the sample. For example, RNA is much more sensitive to temperature changes than genomic DNA, as Beaver says: People have dug up skeletons from years ago and still managed to sequence all the DNA, whereas RNA will degrade if its in a 80C freezer that's been opened too many times! This poses a problem for freezing samples if you dont yet know how you plan to use them.
If you've got very limited tissue, you've got to decide what the best way is to preserve this material as analysis methods improve, says Iqbal. I remember when I started out probably 18 years ago, we would get very large lymph node biopsies with more tissue than we could possibly use so were able to preserve in multiple ways, whereas now core biopsies are prevalent for diagnosis and so you've got very limited material to preserve for research purposes.
The Basics of Cellular Cryopreservation
Cryopreservation has become a routine practice in biomedical research and clinical medicine. When frozen and kept properly, specimens may remain in a state of suspended cellular metabolism indefinitely and can be thawed as needed. To reap the benefits of this process, it is important to have a thorough understanding of the key aspects of cryopreservation. Download this guide to the basics of cellular cryopreservation for research and clinical use.
Sponsored Content
DMSO-based cryopreservation is a great method, but we really need to think about the efficiency of things, agrees Gibson. For a lot of cell types we normally freeze using standard methods we might only get between 20 and 60% of those cells back after freezing. If we can get that number higher, then we can either remove smaller batches from the freezer, or if using rare cells or primary cells which are harder to culture and expand, then the more you recover the faster you can do your research, or the more experiments you can do per batch.
To address this need, Gibson is researching biomimetic materials, and one of the proteins theyve been working to mimic is called the antifreeze protein, or an ice-binding protein.3 These are proteins that somehow manage to selectively bind to ice. They can tell the difference between liquid water and frozen water- it's quite remarkable. These ice-binding proteins are produced by a huge range of species, most famously fish found at the Earths poles. The proteins bind ice crystals to stop them growing so that the fish can survive at lower temperatures than fish without the proteins. It turns out there's quite a few proteins and polysaccharides which either make or stop ice forming or control how and when it forms. If we can mimic that with synthetic polymers, then we could change how we freeze cells inspired by how nature protects itself during cold. The team has already shown that they can use the polymers to reduce the amount of cryoprotectant while freezing bacterial cells.4
Another advance would be to be able to freeze cells already attached to tissue culture plates, says Gibson. With cells in suspension, you have to put them onto plates and let them grow before you can use them. We are interested in how we can freeze cells on those plates so that you can take them out of the freezer and they're ready to use. Gibsons lab has developed some materials that work not by affecting the ice, but by effectively protecting cells during this freezing process.5 We find these really remarkable levels of cell recovery. One of our most exciting results was by adding in polyampholyte to cells in monolayers, attached to tissue culture plastic, and we saw a dramatic increase from < 20% to > 80% of the cells being recovered.6
This research into the science of freezing isnt just limited to life sciences either its being applied to everything from making ice cream taste creamier with less fat to avoiding freezethaw damage to concrete.7
If you can make research any more efficient, that's a great outcome, but research like this is also helping to address basic questions about how these low temperatures are affecting life and how we can store things better, and more efficiently.
References
1. Tavukcuoglu S, Al-Azawi T, Khaki AA, Al-Hasani S, et al. Is vitrification standard method of cryopreservation. Middle East Fertil. Soc. J. 2012;17:152156. doi: 10.1016/j.mefs.2012.07.007.
2. Awan M, Buriak B, Fleck R, et al. Dimethyl sulfoxide: a central player since the dawn of cryobiology, is efficacy balanced by toxicity? Regenerative Medicine 2020; 15 (3) doi: 10.2217/rme-2019-0145
3. Carpenter JF and Hansen TN. Antifreeze protein modulates cell survival during cryopreservation: mediation through influence on ice crystal growth. Proc Natl Acad Sci USA. 1992;89(19):89538957. doi: 10.1073/pnas.89.19.8953
4. Hasan M, Fayter AER, Gibson MI. Ice recrystallization inhibiting polymers enable glycerol-free cryopreservation of microorganisms. Biomacromolecules 2018;19(8):33713376. doi: 10.1021/acs.biomac.8b00660
5. Stubbs C, Bailey TL, Murray K, Gibson M. Polyampholytes as emerging macromolecular cryoprotectants. Biomacromolecules 2020;21(1):717. doi: 10.1021/acs.biomac.9b01053
6. Bailey TL, Stubbs C, Murray K, Toms RMF, Otten L, Gibson MI. Synthetically scalable poly(ampholyte) which dramatically enhances cellular cryopreservation. Biomacromolecules 2019;20(8):31043114. doi: 10.1021/acs.biomac.9b00681
7. Frazier SD, Matar MG, Osio-Norgaard J, Aday AN, Delesky EA, Srubar WV. Inhibiting freeze-thaw damage in cement paste and concrete by mimicking natures antifreeze. Cell Reports Physical Science 2020;1:100060. doi: 10.1016/j.xcrp.2020.100060.
Continued here:
Cryopreservation: Applications and Advances - Technology Networks
- Mesenchymal stem cell based therapies for uveitis: a systematic review of preclinical studies | Eye - Nature.com - April 10th, 2024
- Development of exosome therapy to treat inflammatory bowel disease by VesiCURE Therapeutics - StreetInsider.com - April 10th, 2024
- Adipose Derived Stem Cell Therapy Market Will Show the Highest Growth Rates, Incredible Demand by 2031 - WhaTech - April 3rd, 2024
- Injectable microspheres adhering to the cartilage matrix promote rapid reconstruction of partial-thickness cartilage defects - ScienceDirect.com - March 30th, 2024
- Safety and Potential Effect of Intrauterine Infusion of Autologous Adipose Tissue-Derived Regenerative Cells in ... - Cureus - March 30th, 2024
- Instructing iPS cell-derived mesenchymal stem cells to inhibit abnormal bone formation in FOP - Medical Xpress - March 28th, 2024
- Clinical application of mesenchymal stem cell in regenerative medicine ... - March 22nd, 2024
- Mesenchymal Stem Cells: The Past Present and Future - March 22nd, 2024
- CytoMed Diversifies into Regenerative Medicine after Research Collaboration with Singapore's Sengkang General ... - Yahoo Finance - March 20th, 2024
- Overexpression of Wnt5a promoted the protective effect of mesenchymal stem cells on Lipopolysaccharide-induced ... - BMC Infectious Diseases - March 20th, 2024
- Mesenchymal Stem Cells: What We Have Learned and How to Manage Them - MDPI - March 17th, 2024
- Mesenchymal stem cells and their microenvironment - March 17th, 2024
- Mesenchymal stem/stromal cells as a valuable source for the treatment ... - March 17th, 2024
- Proteomic characterization of hUC-MSC extracellular vesicles and evaluation of its therapeutic potential to treat ... - Nature.com - March 13th, 2024
- The impact understanding of exosome therapy in COVID-19 and preparations for the future approaches in dealing with ... - Nature.com - March 13th, 2024
- CytoMed Therapeutics Limited Announces Research Collaboration with Singapore Sengkang General Hospital to ... - Yahoo Finance UK - March 7th, 2024
- CytoMed Therapeutics Limited Announces Research Collaboration with Singapore Sengkang General Hospital to ... - Yahoo Finance - March 6th, 2024
- Combatting osteoarthritis with cartilage replacement therapy - Drug Target Review - March 3rd, 2024
- Ethical considerations in Stem Cell therapy for ALS - Cyprus Mail - February 27th, 2024
- Therapeutic Solutions International Files Patent on Facilitating Effects of JadiCells on Gene Therapy Mediated Cell ... - Business Wire - February 27th, 2024
- Stem Cell Therapy for Ulcerative Colitis - Health Central - February 27th, 2024
- Expanding the Horizons of Cell and Gene Therapy - RegMedNet - February 24th, 2024
- Early differentiation of mesenchymal stem cells is reflected in their dielectrophoretic behavior | Scientific Reports - Nature.com - February 22nd, 2024
- Orthopaedics Department's Regenerative Research Highlighted at ORS Annual Meeting - InventUM - University of Miami - February 20th, 2024
- Cellular uptake and in vivo distribution of mesenchymal-stem-cell-derived extracellular vesicles are protein corona ... - Nature.com - February 18th, 2024
- New University spin-out developing novel adult stem cell-based Therapies - News - University of Liverpool - News - February 15th, 2024
- Examining the potential of the common bovine as a potential therapeutic research model - Medical Xpress - February 15th, 2024
- Intentional Interference: Genetic Engineering Medium Aids The Transfection Of MSCs With siRNA - BioProcess Online - February 15th, 2024
- Cellular senescence is associated with osteonecrosis of the femoral head while mesenchymal stem cell conditioned ... - Nature.com - February 9th, 2024
- Stem Cell Therapy for Crohns Disease Shows Promising Results - RegMedNet - February 9th, 2024
- Targeted transcriptomic analysis of synovial tissues from horses with septic arthritis treated with immune-activated ... - American Veterinary Medical... - February 7th, 2024
- Mesenchymal Stem Cell Immunomodulation: Mechanisms and ... - Cell Press - February 7th, 2024
- New Cell Therapy for ARDS: A Groundbreaking Development and Other Respiratory Health Breakthroughs - Medriva - February 7th, 2024
- Effective treatment of optic neuropathies by intraocular delivery of MSC-sEVs through augmenting the G-CSF ... - pnas.org - January 31st, 2024
- Global Stem Cell Therapy Industry Outlook to 2028, Driven by Therapeutic Innovations and Clinical Advancements ... - Yahoo Finance - January 29th, 2024
- Out-of-this-world study will test how stem cells function in space - YP - January 29th, 2024
- The Applications of Cell Therapy - Technology Networks - January 26th, 2024
- STEM CELL THERAPY FOR MS COST: WHAT YOU NEED TO KNOW? - Island Echo - January 24th, 2024
- Understanding Neuromyelitis Optica: Role of NF-B and Therapeutic Potential - Medriva - January 20th, 2024
- Faculty member Arnold Caplan passes away The Daily The Daily - The Daily | Case Western Reserve University - January 18th, 2024
- Effects of fine particulate matter on bone marrow-conserved hematopoietic and mesenchymal stem cells: a systematic ... - Nature.com - January 11th, 2024
- Expression of HLA-DR by mesenchymal stromal cells in the platelet lysate era: an obsolete release criterion for MSCs ... - Journal of Translational... - January 11th, 2024
- NurOwn and its exosomes for ALS given patents in Europe, elsewhere - ALS News Today - January 8th, 2024
- Single-cell analysis reveals the stromal dynamics and tumor-specific characteristics in the microenvironment of ovarian ... - Nature.com - January 8th, 2024
- First in Class Combination Stem Cell and Exosome Therapy to Treat Pulmonary Fibrosis in Long Haul Covid Patients ... - Yahoo Finance - January 4th, 2024
- Choosing the Right Excipients for MSC and iPSC Therapies - Pharmaceutical Technology Magazine - January 4th, 2024
- Mitochondria Transplantation Therapy It's Farther Along Than You Think - BioProcess Online - January 2nd, 2024
- Embryonic-stem-cell-derived mesenchymal stem cells relieve experimental contact urticaria by regulating the functions ... - Nature.com - December 21st, 2023
- Type I collagen and fibromodulin enhance the tenogenic phenotype of hASCs and their potential for tendon ... - Nature.com - December 19th, 2023
- Could the 'central dogma' of biology be misleading bioengineers? - Phys.org - December 13th, 2023
- The Risks of Stem Cell and Exosome Treatments for Long COVID: A Call for Stricter Regulations - Medriva - December 13th, 2023
- Mesenchymal stem cells from biology to therapy - PMC - December 11th, 2023
- The Safety and Efficacy of Human Umbilical Cord-Derived ... - Cureus - December 5th, 2023
- The pivotal role of Nrf2 signal axis in IDD | JIR - Dove Medical Press - December 5th, 2023
- Futuristic Novel Therapeutic Approaches in the Treatment of ... - Cureus - December 3rd, 2023
- Cartilage's Contribution in Otology: A Comprehensive Review of Its ... - Cureus - December 3rd, 2023
- Mesenchymal stem or stromal cells: a review of clinical applications ... - November 29th, 2023
- Comparative evaluation of 3D-printed and conventional implants in ... - Nature.com - November 29th, 2023
- Expression of E-cadherin and N-cadherin in Epithelial-to ... - Cureus - November 29th, 2023
- Enhancing the immunosuppressive properties of human umbilical cord mesenchymal stem cells - Phys.org - November 29th, 2023
- 'Great Potential' in Stem Cell Therapies for Knee Osteoarthritis ... - Pain News Network - November 25th, 2023
- Dietary Sources, Bioavailability, and Functions of Ascorbic Acid ... - Cureus - November 25th, 2023
- FY 2024 VA-Funded Projects - VA's Office of Research and Development - November 25th, 2023
- Real-world data suggest effectiveness of the allogeneic ... - Journal of Translational Medicine - November 23rd, 2023
- Mesenchymal Stem Cells: Characteristics, Function, and Application - November 23rd, 2023
- Combined application of therapeutic viruses and nanomaterials to ... - News-Medical.Net - November 23rd, 2023
- Progress in the Development of Stem Cell-Derived Cell-Free ... - Dove Medical Press - November 23rd, 2023
- Improving the therapeutic efficacy of oncolytic viruses for cancer ... - Journal of Translational Medicine - November 23rd, 2023
- 11.22.23 -- How To Lose A Batch In 10 Days - BioProcess Online - November 23rd, 2023
- The Pros and Cons of Mesenchymal Stem Cell-Based Therapies - November 19th, 2023
- Leptin's influence on MMP-1 expression | JIR - Dove Medical Press - November 15th, 2023
- TWIST1 and TSG6 are coordinately regulated and function as ... - Science - November 11th, 2023
- Association between severe acute malnutrition in childhood and ... - BioMed Central - November 11th, 2023
- Global Market for Treatments for Syndromes of Progressive Ataxia and Weakness Disorders - Yahoo Finance - November 11th, 2023
- Stem Cell Restore Reviews - Does It Work? Real Green Valley ... - The Daily World - November 11th, 2023
- Biological and genetic characterization of a newly established ... - Nature.com - November 11th, 2023
- Placental Stem Cell Collection and Storage Industry Scope ... - Argyle Report - November 9th, 2023
- In Vitro Evaluation of Light-Induced Cytotoxic Property: Synergistic ... - Cureus - November 9th, 2023
- UF professor launches nonprofit to equalize cell-based therapy access - Mainstreet Daily News Gainesville - November 7th, 2023
- Who are the leading innovators in cell immunomodulation for the ... - Pharmaceutical Technology - November 7th, 2023
Recent Comments